In these days, a colorimetric assay method using an enzyme has been widely used as one of methods for measuring a living body component in a body fluid (specimen) including blood or urine. These methods are introduced into a measuring kit, an automatic analyzer, and a Dry Chemistry test instrument etc., and used in a number of daily clinical examinations (for example, see JP-A-2004-223115).
As one of these measuring methods, there is a quantitation method which comprises having a specific oxidase (oxidative enzyme) acted on a substance to be measured to generate hydrogen peroxide, changing the hydrogen peroxide into active oxygen by peroxidase, oxidizing a coloring reagent with the active oxygen to generate a pigment, and colorimetrically determine the generated pigment.
As the enzyme to be used therein, for example, glucose oxidase has been used for measuring glucose, cholesterol oxidase has been used for measuring cholesterol, uricase has been used for measuring uric acid, and pyruvate oxidase has been used for measuring pyruvic acid. Since each of these enzymes specifically oxidizes only a substrate of the subject to be measured, only subject to be measured can be exclusively determined from a specimen including various components.
In addition, as the coloring reagent, pigments or precursors thereof (chromogen) has been used, in which absorption wavelength characteristics or absorption intensity thereof change by being oxidized with active oxygen. Specifically, amount of subject to be measured can be quantitatively determined by measuring color varying depending on the amount of active oxygen. These pigments and chromogen include those functioning in only one molecule form, and those functioning in a coupled form of different two molecules.
Among them, as the pigment functioning in one molecule form, leuco type pigment has been used. The compound includes benzidine, o-tolidine, o-dianisidine, 2,2′-amino-bis(3-ethylbenzothiazolinone-6-sulfonic acid) (ABTS), bis-(4-diethylamino)-2-sulfophenylmethane (BSPM), bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA), 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP), 3,3′,5,5′-tetramethylbenzidine (TMBZ), bis[4-(N-alkyl-N-sulfopropyl)-2,6-dimethylphenyl]methane (Bis-MAPS), N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotrip henylmethane (TMP), or the like.
Also, as a typical example using the pigment functioning in two molecules form, a method which comprises oxidatively coupling a coupler compound with Trinder's reagent has been known (for example, see: Trinder, P., Ann. Clin. Biochem., 6, 24, 1969, and Barham, D. and Trinder, P., Analyst (London), 97, 142, 1972).
As the coupler compound, 4-aminoantipyrine (4-AA), vanillin diamine sulfonic acid, methylbenzothiazolinone hydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH), aminodiphenylamine, or the derivatives thereof has been used.
As the Trinder's reagent, for example, phenol derivative or aniline derivative has been used. Examples of the phenol derivative include phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, 2,4-dibromophenol, 2,6,4-trichlorophenol, 2,6,4-tribromophenol, 3,5-dichloro-2-hydroxybenzene sulfonic acid, 3-hydroxy-2,4,6-triiodobenzoic acid, or the like. Examples of the aniline derivative include N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-ethyl-N-(3-methylphenyl)-3-acetylethylenediamine (EMAE), N-ethyl-N-(3-methylphenyl)-N-succinylethylenediamine (EMSE), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-(2-carboxyethyl)-N-ethyl-3-methylaniline (CEMB), N,N-bis-(4-sulfobutyl)-3-methylaniline (TODB), N-ethyl-N-(2-succinylaminoethyl)-3-methylaniline (ESET), N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HSDA), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), or the like.
In practical products, a pigment functioning in two molecules form has a tendency to be employed much more, because a pigment functioning in one molecule form may have less stability when used in a reagent composition. In addition, among the pigments functioning in two molecules form, 4-aminoantipyrine (4-AA), which is most stable among the above-described compounds, has been frequently employed as a coupler compound, and aniline derivatives have been employed much more as a Trinder's reagent, because these compounds are more advantageous in respect to stability, coloring intensity and wave length, as compared with highly volatile phenol derivatives.